The advent of ultrashort (femtosecond) laser light pulses as laboratory tool opens up new opportunities to probe and manipulate anatomy and function in nervous systems. Ultrashort pulses are the essential means to drive the nonlinear absorption of light by biomolecules, which leads to a localized region of excitation and forms the basis of two-photon microscopy. More recently, high-fluence ultrashort pulses have been exploited to reliably create micron-sized ablations in brain tissue with minimal collateral damage. These ablations are the driving technology in an all-optical histology, which allows anatomy to be imaged with micrometer resolution throughout the brain. These ablations can also be used to perturb neocortical blood flow as a means to probe normal and diseased tissues. Yet much additional effort is required to use and advance the mixture of multiphoton ablation and imaging techniques as a means to enable studies of neuronal and vascular architectonics. Our proposed instrumentation concerns the use of ultrashort laser pulses for focal ablation at high fluence (energy per area), and imaging, at lower fluence, to address open issues in neuroanatomy and neurovascular coupling. Two synergistic applications will serve as test beds for our technical goals: Establish all-optical based histology as a standard anatomical tool. This includes the optimization of parameters and the correction of spherical aberration for both ablation and imaging. We proposed to reconstruct neuronal and non-neuronal soma and vasculature positions throughout rat vibrissa sensory cortex and to reconstruct mitochondrial density and vasculature throughout vibrissa sensory cortex. Advance the optical induction and monitoring of targeted vascular blocks. We will perturb blood flow in connective arteriol networks as well as deep capillary networks to study flow dynamics in different angioarchitectures. The proposed advancement in the breadth of nonlinear optical methods will provide a novel tool for manipulating and probing tissues that is simultaneously imaged with two-photon microscopy. We will make these tools reliable and readily available to the biomedical community. The proposed model systems may substantially improve upon our understanding of brain architectonics and stroke formation. That may lead to improvements in preclinical models to assay therapeutics for stroke.